RAB26 is important in the regulation of membrane trafficking and cell motility. Recently, RAB26 has received increasing attention in cancer research. However, the functional role of RAB26 in prostate cancer (PCa) remains to be elucidated. Single-cell RNA sequencing data (GSE141445) analysis indicated that RAB26 was widely expressed in PCa cells, especially in luminal cells and basal cells. High RAB26 expression in patients was found to be significantly associated with advanced pathological stage, Gleason score, and poor prognosis. Furthermore, our experimental results showed that RAB26 promoted the proliferation, migration, and invasion of PCa cells, as well as influenced the stemness of PCa cells in vitro. Besides, the transcription sequence indicated that RAB26 might promote the metastatic potential of PCa by promoting epithelial–mesenchymal transition through cascades of MAPK/ERK pathways. Finally, we found that RAB26 activated TWIST1 expression and consequently induced epithelial–mesenchymal transition, based on its interaction with the TWIST1 promoter. In conclusion, RAB26 promotes the aggressive progression of PCa and stemness of tumor cells, which is an independent biomarker for the prognosis of PCa.